This invention relates to a novel method for the production of a blood coagulation inhibitor known as tissue factor pathway inhibitor (TFPI) and alternatively known as lipoprotein associated coagulation inhibitor (LACI).
Plasma contains a multivalent Kunitz-type inhibitor of coagulation, referred to herein as tissue factor pathway inhibitor (TFPI). This name has been accepted by the International Society on Thrombosis and Hemostasis, Jun. 30, 1991, Amsterdam. TFPI was first purified from a human hepatoma cell, Hep G2, as described by Broze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987), and subsequently from human plasma as reported by Novotny et al., J. Biol. Chem. 264, 18832-18837 (1989); Chang liver and SK hepatoma cells as disclosed by Wun et al., J. Biol. Chem. 265, 16096-16101 (1990). TFPI cDNA have been isolated from placental and endothelial cDNA libraries as described by Wun et al., J. Biol. Chem. 263, 6001-6004 (1988); Girard et al., Thromb. Res. 55, 37-50 (1989). The primary amino acid sequence of TFPI, deduced from the cDNA sequence, shows that TFPI contains a highly negatively charged amino-terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. The first Kunitz-domain of TFPI is needed for the inhibition of the factor VII.sub.a /tissue factor complex and the second Kunitz-domain of TFPI is responsible for the inhibition of factor X.sub.a according to Girard et al., Nature 328, 518-520 (1989), while the function of the third Kunitz-domain remains unknown. See also copending application Ser. No. 07/301,779, filed Jan. 26, 1989, now allowed. TFPI is believed to function in vivo to limit the initiation of coagulation by forming an inert, quaternary factor X.sub.a : TFPI: factor VII.sub.a : tissue factor complex. Further background information on TFPI can be had by reference to the recent reviews by Rapaport, Blood 73, 359-365 (1989); Broze et al., Biochemistry 29, 7539-7546 (1990).
Recombinant TFPI has been expressed as a glycosylated protein using mammalian cell hosts including mouse C127 cells as disclosed by Day et al., Blood 76, 1538-1545 (1990), baby hamster kidney cells as reported by Pedersen et al., J. Biol. Chem. 265, 16786-16793 (1990), Chinese hamster ovary cells and human SK hepatoma cells. The C127 TFPI has been used in animal studies and shown to be effective in the inhibition of tissue factor-induced intravascular coagulation in rabbits according to Day et al., supra, and in the prevention of arterial reocclusion after thrombolysis in dogs as described by Haskel et al., Circulation 84, 821-827 (1991).
The cloning of the TFPI cDNA which encodes the 276 amino acid residue protein of TFPI is further described in Wun et al., U.S. Pat. No. 4,966,852, the disclosure of which is incorporated by reference herein.